Antagonists of MCP proteins

ABSTRACT

Novel antagonists of MCP proteins, in particular of MCP-1 protein, can be obtained by generating MCP mutants whose GAG binding site, located at the N-terminal of MCP proteins, is eliminated following non-conservative substitutions. Compounds prepared in accordance with the present invention can be used in the treatment or prevention of diseases related to an undesirable activity of MCP proteins such, such as inflammatory disease, autoimmune diseases, vascular diseases, and cancer.

CROSS-REFERENCE TO RELATED APPLICATION

This application is the U.S. national stage application of InternationalPatent Application No. PCT/EP03/50097, filed Apr. 9, 2003, which claimsthe benefit of U.S. Provisional Patent Application No. 60/371,442, filedApr. 10, 2002.

FIELD OF THE INVENTION

The present invention is directed to novel antagonists of MCP proteins,and in particular of human MCP-1, which have been generated byappropriately mutagenising MCP proteins.

BACKGROUND OF THE INVENTION

Chemokines are small secreted pro-inflammatory proteins, which mediatedirectional migration of leukocytes from the blood to the site ofinjury. Depending on the position of the conserved cysteinescharacterizing this family of proteins, the chemokine family can bedivided structurally in C, C-C, C-X-C and C-X₃-C chemokines, to whichcorresponds a series of membrane receptors (Baggiolini M et al., 1997;Fernandez E J and Lolis E, 2002). Usually chemokines are produced at thesite of injury and cause leukocyte migration and activation, playing afundamental role in inflammatory, immune, homeostatic and angiogenicprocesses. These molecules, therefore, offer the possibility fortherapeutic intervention in diseases associated to such processes, inparticular by inhibiting specific chemokines and their receptors at thescope to preventing the excessive recruitment and activation ofleukocytes (Baggiolini M, 2001; Loetscher P and Clark-Lewis I, 2001;Godessart N and Kunkel S L, 2001).

Monocyte chemoattractant protein 1 (from now on, MCP-1) is a member ofthe CC chemokine family also known under various names such as CCL2,Small Inducible Cytokine A2 (SCYA2), Monocyte Chemotactic And ActivatingFactor (MCAF), Monocyte Secretory Protein Je, Monocyte ChemotacticFactor, and HC11. This chemokine is capable of promoting the recruitmentof monocytes and basophils in response to injury and infection signalsin various inflammatory diseases, different types of tumors, cardiacallograft, AIDS, and tuberculosis (Gu L et al., 1999).

Structurally and functionally homologous proteins have been identifiedand called MCP-2 (CCL7), MCP-3 (CCL8), MCP-4 (CCL13), and Eotaxin(CCL11). This subfamily of C-C chemokines is significantly distinct fromother C-C chemokines, such as RANTES or MIP-1alpha/beta, and probablycoevolved from a common progenitor sequence. They have a similarreceptor usage, binding in particular CCR2 (but also for CCR1, CCR3, andCCR5). Therefore, many of the immunological and inflammatory agonisticor antagonistic activities of these C-C chemokines are common (Hughes AL and Yeager M, 1999; Berhkout T A et al., 1997; Luster A D andRothenberg M E, 1997, Proost P et al., 1996).

The physiological activities associated with MCP-1 have been extensivelystudied by means of transgenic animals and other animal models, whichdemonstrate that MCP-1 controls recruitment of monocytes and of othercell types (astrocytes, for example) in many infectious, inflammatoryand autoimmune diseases, as well as the expression of cytokines relatedto T helper responses. Other diseases that appear induced by MCP-1 arevascular disorders (restenosis after coronary intervention,arteriosclerosis, atherosclerosis, ischemia, stroke) and cancer-relatedangiogenesis (Ikeda Y et al., 2002; Egashira K et al., 2002; Gu L etal., 2000; Salcedo R et al., 2000; Gosling J et al, 1999; Lu B et al,1998; Rutledge B J et al., 1995).

Since MCP-1 targeting is considered as a possible therapeutic approachfor several diseases, different types of MCP-1 antagonists have beendescribed in the literature, obtaining more or less important inhibitoryeffect on MCP-1-induced pathological activities (Dawson J, 2003).Examples of MCP-1 antagonists are an N-terminal deletion mutant ofMCP-1, natural or synthetic, missing the N-terminal amino acids 2 to 10(Egashira K et al., 2000; Zhang Y and Rollins B J, 1995; McQuibban G Aet al., 2002), anti-MCP-1 monoclonal antibodies (Ajuebor M N et al.,1998; Eghtesad M et al., 2001), RNA aptamers (Rhodes A et al., 2001),peptides designed on sequences internal to MCP-1 (Reckless J andGrainger D J, 1999), MCP-1 antagonists peptide mimics (Kaji M et al.,2001), antisense oligonucleotides (WO 94/09128), small molecules(Mirzadegan T et al., 2000), polymer-modified MCP-1 (WO 02/04015), orviral decoy receptors (Alexander J M et al., 2002; Beck C G et al.,2001).

Structurally, MCP proteins present a N-terminal loop and three β-sheetsoverlaid by a α-helix at the C-terminal end (Handel T M et al., 1996;Lubkowski J, et al., 1997; Blaszczyk J et al., 2000). The literatureprovides many examples of structure-activity studies (Gong J H andClark-Lewis 1, 1995; Zhang et al., 1996; Beall C J et al., 1996; SteitzS A et al., 1998; Gu L et al., 1999; Hemmerich S et al., 1999; Seet B Tet al., 2001) in which MCP-1 mutants have reduced activity and/oraffinity for the receptor or other binding proteins have been obtainedby expressing N-terminal truncations (as in many other chemokines), orsingle mutations at residues 3, 8, 10, 13, 15, 18, 19, 24, 28, 30, 37,38, and 39 (following the numbering of mature human MCP-1). Similarresults have been obtained for Eotaxin (Mayer M R and Stone M J, 2001).

Chemokines interact with proteoglycans (PGs) and glycosaminoglycans(GAGs) a feature common to many cell-signaling soluble molecules(interleukins, growth factors). Proteoglycans are negatively chargedproteins that are post-translationally modified by the addition ofglycosaminoglycan side chains at serine residues. Clusters of basicresidues (mainly Arginines and Lysines) allow proteins to interact withGAGs, which commonly are characterized by the disaccharide repeats suchas heparin, chondroitin sulfate, heparan sulfate, dermatan sulfate, andhyaluronic acid). PGs and GAGs can be present on membrane surfaces aswell as soluble molecules, probably at the scope to protect thismolecule from proteolysis in the extracellular environment. It has beenalso proposed that GAGs may help the correct presentation of cellsignaling molecules to their specific receptor and, eventually, also themodulation of target cell activation. In the case of chemokines, theconcentration into immobilized gradients at the site of inflammationand, consequently, the interaction with cell receptors and theiractivation state seem to be modulated by the specific GAG. Theinteraction with GAGs and the formation of these gradients have beenclearly demonstrated for many chemokines, including MCP-1, measuring therelative affinity. Therefore, it has been suggested that the modulationof such interactions may represent a therapeutic approach ininflammatory disease (Hoogewerf A J et al., 1997; Kuschert G et al.,1999; Ali S et al., 2001; Patel D et al., 2001; WO 02/28419; WO99/50246).

However, the structural requirements and functional effects of GAG/MCP-1interactions have been poorly studied. It is known that GAGs canmodulate the activity and production of MCP-1 secreted from endothelialcells (Douglas M S et al., 1997). It has been also reported thatsubstitution of Lysine 58 and Histidine 66 with Alanines in theC-terminal of MCP-1, prevents GAG binding without affecting receptorbinding, Ca²⁺ influx, or chemotactic activity (Chakravarty L et al,1998), but there is no disclosure in the prior art of which may be otherGAG binding sites of MCP-1, and which in vivo effects can be consequentto their elimination. Even though extensive studies have been performedon some chemokines, it is not possible to anticipate, on the basis ofthe sequence homology, which residues have to be modified withnon-conservative substitutions to impair GAG binding, and which effectscan be obtained, since there is a significant structural diversity ofGAG binding domains in chemokines (Lortat-Jacob H et al., 2002).

SUMMARY OF THE INVENTION

It has been found that a dibasic site at the N-terminal of human MCP-1(Arginine 18, Lysine 19) is responsible for the interaction of MCP-1with GAGs. The elimination of this site by non-conservativesubstitutions (for example, with Alanines) allows to generate MCP-1mutants having not only have a reduced tendency to interact with GAG,but a surprising in vivo, dose-related antagonistic activities on MCP-1.Such evidence can be exploited to use mutants of MCP-1, and of other MCPproteins, as antagonists of the corresponding MCP protein. Compoundsprepared in accordance with the present invention can be used to inhibitthe migration and activation of leukocytes expressing their receptors,thereby providing useful therapeutic compositions for use in thetreatment of diseases related to excessive or uncontrolled leukocytemigration, such as inflammation and autoimmune diseases. Other featuresand advantages of the invention will be apparent from the followingdetailed description.

DESCRIPTION OF THE FIGURES

FIG. 1: (A) amino acid sequences of human and mutated MCP-1 proteins asexpressed and tested in the Examples (mutated amino acids areunderlined). The N-terminal methionine in MCP-1WT* and MCP-1WT*2A wasremoved during purification by aminopeptidase treatment to avoid anyinterference on the activity of the protein due to this addition alresidue. (B) Alignment of the mature forms of human MCP-1 (CCL2;SWISSPROT Acc. No. P13500), MCP-2 (CCL7; SWISSPROT Acc. No. P80075),MCP-3 (CCL8; SWISSPROT Acc. No. P80098), MCP4 (CCL8; SWISSPROT Acc. No.Q99616), and Eotaxin (CCL11, SWISSPROT Acc. No. P51671). The basicresidues identified in MCP-1 in the present patent application as beinginvolved in the binding with GAGs (residues 18 and 19) and the conservedcorresponding residues in the more homologous human proteins are boxed.Other basic residues conserved amongst all human MCP proteins areunderlined.

FIG. 2: graph representing the results of the heparin binding assayperformed with [³H]-heparin and, as chemokine, either MCP-1WT* (◯) orMCP-1WT*2A (●).

FIG. 3: graph representing the results of the equilibrium competitionreceptor binding assay performed by monitoring the displacement of[¹²⁵I]-MCP-1 from CCR2-expressing CHO membranes consequent to theaddition, as chemokine, of hMCP-1 (□), MCP-1WT* (◯), or MCP-1WT*2A (●).

FIG. 4: graph representing the results of the transwell chemotaxis assayperformed using THP-1 cells and, as chemotactic agent, recombinant humanMCP-1 (□), MCP-1WT*, (◯) or MCP-1WT*2A (●).

FIG. 5: graph summarizing the results of the peritoneal cellsrecruitment assay, performed in mice using MCP-1WT* and/or MCP-1WT*2A.The concentrations of MCP-1WT*2A showing a statistically significantinhibition activity on the number of cells recruited by MCP-1WT* areindicated in the graph with *.

FIG. 6: graph summarizing the results of the delayed contacthypersensitivity assay. Mice were treated with 0.5 mg/kg MCP-1WT*2A (▪)or with vehicle only (□). The effect is measured in terms of earswelling volume each day during treatment.

FIG. 7: graph summarizing the effects on body weight of mice receivingbleomycin for inducing lung fibrosis. The average weight of control mice(□) is compared with the average weight of treated mice receiving aswell an intraperitoneal administration of 0.25 mg/kg MCP-1WT*2A (●) orPBS only (◯). The indicated weight is an average weight value for eachgroup of mice.

FIG. 8: graph comparing the fibrosis levels in untreated andbleomycin-treated mice, which were additionally treated with PBS only,or with an intraperitoneal administration of 0.25 mg/kg MCP-1WT*2A.Fibrosis levels were measured either spectroscopically (top) orhistologically (bottom) as described in the Examples.

FIG. 9: graph comparing the clinical score measured in ExperimentalAutoimmune Encephalomyelitis (EAE) animal models treated with PBS only(Group 1), 1 microgram/mouse of MCP-1WT*2A (Group 2), or 10micrograms/mouse of MCP-1WT*2A (Group 3).

FIG. 10: graph comparing the clinical score measured in collagen inducedarthritis (CIA) animal models treated with PBS only, or 1microgram/mouse of MCP-1WT*2A.

FIG. 11: graph comparing the amount of cells isolated from trachea ofanimal models treated and challenged with PBS only (Group 1), treatedwith PBS and challenged with ovalbumin (Group 2), or treated with 10microgram/mouse of MCP-1WT*2A and challenged with ovalbumin (Group 3).

DETAILED DESCRIPTION OF THE INVENTION

In view of the literature mentioned above, there is no indication that aspecific dibasic site in the N-terminus of human MCP-1 defines a GAGbinding site, and that the non-conservative substitution of the residuesin this site leads to molecules having antagonistic activity on MCP-1.Moreover, given the conservation of this dibasic site amongst known MCPproteins, as well as of other residues which are basic and/or known tobe involved in GAG binding, it can be inferred that the antagonists ofMCP proteins can be obtained by non-conservative substitutions in theresidues corresponding to the ones functionally characterized in humanMCP-1.

The main object of the present invention is to provide novel antagonistsof MCP proteins consisting of mutants of MCP proteins in which thefollowing combinations of residues, numbered on the sequence of humanmature MCP-1, are substituted to Alanine, Glycine, Serine, Threonine,Proline, Aspartic acid, Asparagine, Glutamic acid, or Glutamine:

a) 18 and 19;

b) 18 and/or 19, together with 58;

c) 18 and/or 19, together with 66;

d) 18 and/or 19, together with 58 and 66;

e) 18 and/or 19, together with one or more of the following: 24, 44, 49,75.

The present patent application provides surprising in vivo and in vitrodata obtained with a novel recombinant MCP-1 mutant in which Arginine 18and Lysine 19 were substituted with Alanines, which is a particularexample of the combinations described above. These results, combinedwith the knowledge on the sequence and the structure of other highlyconserved MCP proteins suggests that this dibasic site can play not onlya general role in MCP proteins biological activity, but also can bemodified accordingly in these homologous proteins to obtain antagonistmolecules.

The basic residues which have to be mutated in a non-conservative mannerin MCP proteins to obtain molecules having antagonistic properties areessentially both residues in positions 18 and 19, at least one of thebasic residues in position 18 and 19 combined with at least one of theresidues already known to be involved in GAG binding, such as 58 and 66(Chakravarty L et al., 1999), or at least one of the basic residues inposition 18 and 19 combined with at least one of the other basicresidues which are conserved in all human MCP proteins (FIG. 1B;Berhkout T A et al., 1997). The amino acid replacing the basic residueis preferably a non-polar, small amino acid like Alanine or Glycine, butother amino acids are appropriate, provided that they have a charge anddimension which poorly interfere with the structure of the protein and,at the same time, are incompatible with GAG binding, for example Serine,Threonine, Proline, Aspartic acid, Asparagine, Glutamic acid, orGlutamine.

Therefore the main object of the present invention is to provide mutantsof MCP proteins which contain a combination of the mutations definedabove, and which act as antagonists of MCP proteins.

The term “antagonist of MCP proteins” means any molecule, which acts asantagonist to the corresponding mature full-length, naturally-occurring(wild-type) MCP protein. MCP-1 antagonists known in the art involvesmodifications

In the sense of the present application, the term MCP proteins includehuman MCP-1, human MCP-2, human MCP-3, human MCP4, and human Eotaxin(FIG. 1B; the legend indicates the corresponding SWISSPROT accessionnumbers), as well as any other protein having at least 70%, preferably80%, and more preferably 90% of homology with human mature MCP-1, MCP-2,MCP-3, MCP-4, or Eotaxin and containing a basic, positively chargedamino acid (Arginine, Lysine, or Histidine) in all the positionsidentified above.

Further objects of the present invention are antagonists of MCP proteinsselected from:

-   -   a) active mutants of the above defined mutants of MCP proteins        in which one or more amino acid residues have been added,        deleted, or substituted without interfering with the        antagonistic activity;    -   b) peptide mimetics designed on the sequence and/or the        structure of polypeptides or peptides of (a);    -   c) polypeptides or peptides comprising the amino acid sequence        of (a) or (b), and an amino acid sequence belonging to a protein        sequence other than the corresponding MCP protein;    -   d) active fractions, precursors, salts, or derivatives of (a),        (b), or (c).

The antagonistic properties of MCP mutants defined above, andexemplified in the present patent application using MCP-1WT*2A as MCP-1antagonist, can be maintained, or even potentiated, in the activemutants. This category of molecules includes natural or artificialanalogs of said sequence, wherein one or more amino acid residues havebeen added, deleted, or substituted, provided they display the samebiological activity characterized in the present invention at comparableor higher levels, as determined by means known in the art and disclosedin the Examples below.

For example, acceptable substitutions should be directed to residues notinvolved in GAG binding, like the substitution of Methionine 64 with anIsoleucine shown in the examples (MCP-1WT*2A; SEQ ID NO: 3) to improvepurification without altering essential properties of human MCP-1.Another object of the present invention is therefore an MCP-1antagonists having the sequence corresponding to MCP-1WT*2A (SEQ ID NO:3). Alternatively, the MCP-1 antagonists, in addition to thesubstitutions directed to the residues involved to GAG binding, may miss2 to 10 N-terminal amino acids, as in the known N-terminal deletionmutants of MCP-1 (Egashira K et al., 2000; Zhang Y and Rollins B J,1995; McQuibban G A et al., 2002), possibly obtaining improvedantagonistic properties.

Natural analogs are intended the corresponding sequences of MCP proteinsidentified in humans or in other organisms, like mouse MCP-1 (SWISSPROTAcc. No. P10148). Artificial analogs are intended peptides prepared byknown chemical synthesis and/or by site-directed mutagenesis techniques,or any other known technique suitable thereof, which provide a finiteset of substantially corresponding mutated or shortened peptides orpolypeptides which can be routinely obtained and tested by one ofordinary skill in the art using the teachings presented in the prior artand in the Examples of the present patent application.

In accordance with the present invention, preferred changes in theseactive mutants are commonly known as “conservative” or “safe”substitutions, and involve non-basic residues. Conservative amino acidsubstitutions are those with amino acids having sufficiently similarchemical properties, in order to preserve the structure and thebiological function of the molecule. It is clear that insertions anddeletions of amino acids may also be made in the above defined sequenceswithout altering their function, particularly if the insertions ordeletions only involve a few amino acids, e.g., under ten, andpreferably under three, and do not remove or displace amino acids whichare critical to the functional conformation of a protein or a peptide.

The literature provide many models on which the selection ofconservative amino acids substitutions can be performed on the basis ofstatistical and physico-chemical studies on the sequence and/or thestructure of natural protein (Rogov S I and Nekrasov A N, 2001). Proteindesign experiments have shown that the use of specific subsets of aminoacids can produce foldable and active proteins, helping in theclassification of amino acid “synonymous” substitutions which can bemore easily accommodated in protein structure, and which can be used todetect functional and structural homologs and paralogs (Murphy L R etal., 2000). The synonymous amino acid groups and more preferredsynonymous groups are those defined in Table I.

Active mutants produced by substitutions made on the basis of theseteachings, as well as active mutants wherein one or more amino acidswere eliminated or added, are amongst the objects of the presentinvention, that is, novel mutants of MCP proteins having poor GAGbinding properties and antagonistic activity on the corresponding MCPprotein, comparable to the ones of the initially selected mutants, oreven improved if possible.

The above described alternative compounds are intended to comprehendmolecules with changes to the sequence of the mutants of MCP proteinsdefined above which do not affect the basic characteristics disclosed inthe present patent application, particularly insofar as its ability asantagonists is concerned. Similar compounds may result from conventionalmutagenesis technique of the encoding DNA, from combinatorialtechnologies at the level of encoding DNA sequence (such as DNAshuffling, phage display/selection), or from computer-aided designstudies, followed by the validation for the desired activities asdescribed in the prior art and in the Examples below.

Specific antagonists can be obtained in the form of peptide mimetics(also called peptidomimetics) of the MCP mutants above defined, in whichthe nature of peptide or polypeptide has been chemically modified at thelevel of amino acid side chains, of amino acid chirality, and/or of thepeptide backbone. These alterations are intended to provide MCPantagonists having similar or improved therapeutic, diagnostic and/orpharmacokinetic properties.

For example, when the peptide is susceptible to cleavage by peptidasesfollowing injection into the subject is a problem, replacement of aparticularly sensitive peptide bond with a non-cleavable peptide mimeticcan provide a peptide more stable and thus more useful as a therapeutic.Similarly, the replacement of an L-amino acid residue is a standard wayof rendering the peptide less sensitive to proteolysis, and finally moresimilar to organic compounds other than peptides. Also useful areamino-terminal blocking groups such as t-butyloxycarbonyl, acetyl,theyl, succinyl, methoxysuccinyl, suberyl, adipyl, azelayl, dansyl,benzyloxycarbonyl, fluorenylmethoxycarbonyl, methoxyazelayl,methoxyadipyl, methoxysuberyl, and 2,4-dinitrophenyl. Many othermodifications providing increased potency, prolonged activity, easinessof purification, and/or increased half-life are known in the art (WO02/10195; Villain M et al., 2001). Preferred alternative, “synonymous”groups for amino acids included in peptide mimetics are those defined inTable II.

The techniques for the synthesis and the development of peptidemimetics, as well as non-peptide mimetics, are well known in the art(Sawyer T K, 1997; Hruby V J and Balse P M, 2000; Golebiowski A et al.,2001). Various methodology for incorporating unnatural amino acids intoproteins, using both in vitro and in vivo translation systems, to probeand/or improve protein structure and function are also disclosed in theliterature (Dougherty D A, 2000). MCP-1 antagonists peptide mimics areknown in the literature, without being highly homologous to MCP-1 (KajiM et al., 2001).

The present patent application discloses as MCP antagonists polypeptidesor peptides comprising the amino acid sequence as defined above and anamino acid sequence belonging to a protein sequence other than thecorresponding MCP protein. This heterologous latter sequence shouldprovide additional properties without impairing significatively theantagonistic activity, or proving GAG binding properties. Examples ofsuch additional properties are an easier purification procedure, alonger lasting half-life in body fluids, or extracellular localization.This latter feature is of particular importance for defining a specificgroup of fusion or chimeric proteins included in the above definitionsince it allows the molecules defined as MCP antagonists in this patentapplication to be localized in the space where not only where theisolation and purification of these peptides is facilitated, but alsowhere MCP proteins and their receptor naturally interact.

Additional protein sequences which can be used to generate thepolypeptide or peptide of (c) are the ones of extracellular domains ofmembrane-bound protein, immunoglobulin constant region, multimerizationdomains, extracellular proteins, signal peptide-containing proteins,export signal-containing proteins. The choice of one or more of thesesequences to be fused to the mutant of MCP protein is functional tospecific use of said agent. As a general procedure, these fusionproteins can be produced by generating nucleic acid segments encodingthem, using common genetic engineering techniques, and cloning inreplicable vector of viral or plasmid origin which are used to modify aProkaryotic or Eukaryotic host cell, using episomal or non-/homologouslyintegrated vectors, as well as transformation-, infection-, ortransfection-based technologies. These vectors should allow theexpression of the fusion protein including the MCP antagonist in theprokaryotic or eukaryotic host cell under the control of their owntranscriptional initiation/termination regulatory sequences, which arechosen to be constitutively active or inducible in said cell. A cellline substantially enriched in such cells can be then isolated toprovide a stable cell line.

When the additional protein sequence, as in the case of the sequence ofextracellular, export signal, or signal-peptide containing proteins,allows the MCP antagonists to be secreted in the extracellular space,the agent can be more easily collected and purified from cultured cellsin view of further processing or, alternatively, the cells can bedirectly used or administered.

The polypeptides and the peptides of the present invention can be inother alternative forms which can be preferred according to the desiredmethod of use and/or production, for example as active fractions,precursors, salts, derivatives, conjugates or complexes.

The term “active” means that such alternative compounds should maintainthe functional features of the MCP mutants of the present invention, andshould be as well pharmaceutically acceptable and useful.

The term “fraction” refers to any fragment of the polypeptidic chain ofthe compound itself, alone or in combination with related molecules orresidues bound to it, for example residues of sugars or phosphates, oraggregates of the original polypeptide or peptide. Such molecules canresult also from other modifications which do not normally alter primarysequence, for example in vivo or in vitro chemical derivativization ofpeptides (acetylation or carboxylation), those made by modifying thepattern of phosphorylation (introduction of phosphotyrosine, phosphoserine, or phosphothreonine residues) or glycosylation (byexposing the peptide to enzymes which affect glycosylation e.g.,mammalian glycosylating or deglycosylating enzymes) of a peptide duringits synthesis and processing or in further processing steps.

The “precursors” are compounds which can be converted into the compoundsof present invention by metabolic and enzymatic processing prior orafter the administration to the cells or to the body.

The term “salts” herein refers to both salts of carboxyl groups and toacid addition salts of amino groups of the peptides, polypeptides, oranalogs thereof, of the present invention. Salts of a carboxyl group maybe formed by means known in the art and include inorganic salts, forexample, sodium, calcium, ammonium, ferric or zinc salts, and the like,and salts with organic bases as those formed, for example, with amines,such as triethanolamine, arginine or lysine, piperidine, procaine andthe like. Acid addition salts include, for example, salts with mineralacids such as, for example, hydrochloric acid or sulfuric acid, andsalts with organic acids such as, for example, acetic acid or oxalicacid. Any of such salts should have substantially similar activity tothe peptides and polypeptides of the invention or their analogs.

The term “derivatives” as herein used refers to derivatives which can beprepared from the functional groups present on the lateral chains of theamino acid moieties or on the N-/ or C-terminal groups according toknown methods. Such derivatives include for example esters or aliphaticamides of the carboxyl-groups and N-acyl derivatives of free aminogroups or O-acyl derivatives of free hydroxyl-groups and are formed withacyl-groups as for example alcanoyl- or aroyl-groups.

Useful conjugates or complexes of the MCP antagonists of the presentinvention can be generated, using molecules and methods known in the artof the interaction with receptor or other proteins (radioactive orfluorescent labels, biotin), therapeutic efficacy (cytotoxic agents), orimproving the agents in terms of drug delivery efficacy, such aspolyethylene glycol and other natural or synthetic polymers (Pillai Oand Panchagnula R, 2001).

The compounds of the invention may be prepared by any well knownprocedure in the art, including recombinant DNA-related technologiesdescribed above, and chemical synthesis technologies.

Another object of the invention are the DNA molecules comprising the DNAsequences coding for the MCP mutants of the invention, includingnucleotide sequences substantially the same. “Nucleotide sequencessubstantially the same” includes all other nucleic acid sequences that,by virtue of the degeneracy of the genetic code, also code for the givenamino acid sequences.

The invention also includes expression vectors which comprise the aboveDNAs, host cells transformed with such vectors and a process ofpreparation of MCP antagonists of the invention, through the culture inappropriate culture media of said transformed cells, and collecting theexpressed protein.

The DNA sequence coding for the proteins of the invention can beinserted and ligated into a suitable plasmid. Once formed, theexpression vector is introduced into a suitable host cell, which thenexpresses the vector(s) to yield the desired protein.

Expression of any of the recombinant proteins of the invention asmentioned herein can be effected in eukaryotic cells (e.g. yeasts,insect or mammalian cells) or prokaryotic cells, using the appropriateexpression vectors. Any method known in the art can be employed.

For example the DNA molecules coding for the proteins obtained by any ofthe above methods are inserted into appropriately constructed expressionvectors by techniques well known in the art. Double stranded cDNA islinked to plasmid vectors by homopolymeric tailing or by restrictionlinking involving the use of synthetic DNA linkers or blunt-endedligation techniques: DNA ligases are used to ligate the DNA molecules,and undesirable joining is avoided by treatment with alkalinephosphatase.

In order to be capable of expressing the desired protein, an expressionvector should also comprise specific nucleotide sequences containingtranscriptional and translational regulatory information linked to theDNA coding the desired protein in such a way as to permit geneexpression and production of the protein. First in order for the gene tobe transcribed, it must be preceded by a promoter recognizable by RNApolymerase, to which the polymerase binds and thus initiates thetranscription process. There are a variety of such promoters in use,which work with different efficiencies (strong and weak promoters).

For Eukaryotic hosts, different transcriptional and translationalregulatory sequences may be employed, depending on the nature of thehost. They may be derived form viral sources, such as adenovirus, bovinepapilloma virus, Simian virus or the like, where the regulatory signalsare associated with a particular gene which has a high level ofexpression. Examples are the TK promoter of the Herpes virus, the SV40early promoter, the yeast gal4 gene promoter, etc. Transcriptionalinitiation regulatory signals may be selected which allow for repressionand activation, so that expression of the genes can be modulated.

The DNA molecule comprising the nucleotide sequence coding for theprotein of the invention is inserted into vector(s), having the operablylinked transcriptional and translational regulatory signals, which iscapable of integrating the desired gene sequences into the host cell.

The cells that have been stably transformed by the introduced DNA can beselected by also introducing one or more markers allowing for selectionof host cells containing the expression vector. The marker may alsoprovide for phototrophy to an auxotropic host, biocide resistance, e.g.antibiotics, or heavy metals such as copper, or the like. The selectablemarker gene can either be directly linked to the DNA gene sequences tobe expressed, or introduced into the same cell by co-transfection.

Additional elements of the vectors may also be useful for obtaining anoptimal production of proteins of the invention, in particular forselecting a particular cell containing plasmid or viral vector: the easewith which recipient cells, that contain the vector may be recognizedand selected from those recipient cells which do not contain the vector;the number of copies of the vector which are desired in a particularhost; and whether it is desirable to be able to “shuttle” the vectorbetween host cells of different species.

Once the vector(s) or DNA sequence containing the construct(s) has beenprepared for expression the DNA construct(s) may be introduced into anappropriate host cell by any of a variety of suitable means:transformation, transfection, conjugation, protoplast fusion,electroporation, calcium phosphate-precipitation, direct microinjection,etc.

Host cells may be either prokaryotic or eukaryotic. Preferred areeukaryotic hosts, e.g. mammalian cells, such as human, monkey, mouse,and Chinese Hamster Ovary (CHO) cells, because they providepost-translational modifications to protein molecules, including correctfolding or glycosylation at correct sites. Also yeast cells can carryout post-translational peptide modifications including glycosylation. Anumber of recombinant DNA strategies exist which utilize strong promotersequences and high copy number of plasmids that can be utilized forproduction of the desired proteins in yeast. Yeast recognizes leadersequences on cloned mammalian gene products and secretes peptidesbearing leader sequences (i.e., pre-peptides).

After the introduction of the vector(s), the host cells are grown in aselective medium, which selects for the growth of vector-containingcells. Expression of the cloned gene sequence(s) results in theproduction of the desired proteins.

These objects of the invention can be achieved by combining thedisclosure provided by the present patent application on antagonists ofMCP proteins, with the knowledge of common molecular biology techniques.Many reviews (Makrides S C, 1999) and books provides teachings on how toclone and produce recombinant proteins using vectors and Prokaryotic orEukaryotic host cells, such as some titles in the series “A PracticalApproach” published by Oxford University Press (“DNA Cloning 2:Expression Systems”, 1995; “DNA Cloning 4: Mammalian Systems”, 1996;“Protein Expression”, 1999; “Protein Purification Techniques”, 2001).

Examples of chemical synthesis technologies are solid phase synthesisand liquid phase synthesis. As a solid phase synthesis, for example, theamino acid corresponding to the C-terminus of the peptide to besynthetized is bound to a support which is insoluble in organicsolvents, and by alternate repetition of reactions, one wherein aminoacids with their amino groups and side chain functional groups protectedwith appropriate protective groups are condensed one by one in orderfrom the C-terminus to the N-terminus, and one where the amino acidsbound to the resin or the protective group of the amino groups of thepeptides are released, the peptide chain is thus extended in thismanner. Solid phase synthesis methods are largely classified by the tBocmethod and the Fmoc method, depending on the type of protective groupused. Typically used protective groups include tBoc (t-butoxycarbonyl),Cl-Z (2-chlorobenzyloxycarbonyl), Br-Z (2-bromobenzyloxycarbonyl), Bzl(benzyl), Fmoc (9-fluorenylmethoxycarbonyl), Mbh(4,4′-dimethoxydibenzhydryl), Mtr(4-methoxy-2,3,6-trimethylbenzenesulphonyl), Trt (trityl), Tos (tosyl),Z (benzyloxycarbonyl) and Cl2-Bzl (2,6-dichlorobenzyl) for the aminogroups; NO₂ (nitro) and Pmc (2,2,5,7,8-pentamethylchromane-6-sulphonyl)for the guanidino groups); and tBu (t-butyl) for the hydroxyl groups).After synthesis of the desired peptide, it is subjected to thede-protection reaction and cut out from the solid support. Such peptidecutting reaction may be carried with hydrogen fluoride ortri-fluoromethane sulfonic acid for the Boc method, and with TFA for theFmoc method. Totally synthetic MCP proteins are disclosed in theliterature (Brown A et al., 1996).

Purification of the natural, synthetic or recombinant MCP antagonists ofthe invention can be carried out by any one of the methods known forthis purpose, i.e. any conventional procedure involving extraction,precipitation, chromatography, electrophoresis, or the like. A furtherpurification procedure that may be used in preference for purifying theprotein of the invention is affinity chromatography using monoclonalantibodies or affinity groups, which bind the target protein and areproduced and immobilized on a gel matrix contained within a column.Impure preparations containing the proteins are passed through thecolumn. The protein will be bound to the column by heparin or by thespecific antibody while the impurities will pass through. After washing,the protein is eluted from the gel by a change in pH or ionic strength.Alternatively, HPLC (High Performance Liquid Chromatography) can beused. The elution can be carried using a water-acetonitrile-basedsolvent commonly employed for protein purification. The inventionincludes purified preparations of the compounds of the invention.Purified preparations, as used herein, refers to the preparations whichare at least 1%, preferably at least 5%, by dry weight of the compoundsof the invention.

Another object of the present invention is the use of MCP antagonists asabove defined as medicaments, in particular as the active ingredients inpharmaceutical compositions (and formulated in combination withpharmaceutically acceptable carriers, excipients, stabilizers, ordiluents) for treating or preventing diseases related to an undesirableactivity of MCP proteins leading to an excessive migration andactivation of leukocytes expressing their receptors, such as autoimmuneand inflammatory diseases as well as bacterial and viral infections.Non-limitative examples of such diseases are the following: arthritis,rheumatoid arthritis (RA), psoriatic arthritis, psioriasis,osteoarthritis, systemic lupus erythematosus (SLE), systemic sclerosis,scleroderma, polymyositis, glomerulonephritis, fibrosis, lung fibrosis,allergic or hypersensitvity diseases, dermatitis, Type IVhypersensitivity also called delayed-type hypersensitivity or DTH,asthma, chronic obstructive pulmonary disease (COPD), inflammatory boweldisease (IBD), Crohn's disease, ulcerative colitis, multiple sclerosis,septic shock, HIV-infection, transplantation, graft-versus-host disease(GVHD), endometriosis, pancreatitis, thyroiditis, encephalopathies.

In view of the literature on the subject, MCP proteins antagonists canbe as active ingredients in pharmaceutical compositions for thetreatment or prevention of other diseases related to an undesirableactivity of MCP proteins, such as vascular disorders (restenosis aftercoronary intervention, arteriosclerosis, atherosclerosis, ischemia,stroke) or cancer.

Another object of the present invention is, therefore, the method fortreating or preventing any of the above mentioned diseases comprisingthe administration of an effective amount of an MCP protein antagonistof the invention.

The pharmaceutical compositions may contain, in addition to the MCPantagonist, suitable pharmaceutically acceptable carriers, biologicallycompatible vehicles and additives which are suitable for administrationto an animal (for example, physiological saline) and eventuallycomprising auxiliaries (like excipients, stabilizers or diluents) whichfacilitate the processing of the active compounds into preparationswhich can be used pharmaceutically. Such compositions can be eventuallycombined with another therapeutic composition acting synergically or ina coordinated manner with the MCP mutants of the invention. For example,similar synergistic properties of CC-chemokine antagonists have beendemonstrated in combination with cyclosporin (WO 00/16796).Alternatively, the other composition can be based with a compound knownto be therapeutically active against the specific disease (for example,IFN-beta for multiple sclerosis, soluble TNF receptors for rheumatoidarthritis).

The pharmaceutical compositions may be formulated in any acceptable wayto meet the needs of the mode of administration. For example, the use ofbiomaterials and other polymers for drug delivery, as well the differenttechniques and models to validate a specific mode of administration, aredisclosed in literature (Luo B and Prestwich G D, 2001; Cleland J L etal., 2001).

An “effective amount” refers to an amount of the active ingredients thatis sufficient to affect the course and the severity of the disease,leading to the reduction or remission of such pathology. The effectiveamount will depend on the route of administration and the condition ofthe patient.

“Pharmaceutically acceptable” is meant to encompass any carrier, whichdoes not interfere with the effectiveness of the biological activity ofthe active ingredient and that is not toxic to the host to which isadministered. For example, for parenteral administration, the aboveactive ingredients may be formulated in unit dosage form for injectionin vehicles such as saline, dextrose solution, serum albumin andRinger's solution.

Any accepted mode of administration can be used and determined by thoseskilled in the art to establish the desired blood levels of the activeingredients. For example, administration may be by various parenteralroutes such as subcutaneous, intravenous, intradermal, intramuscular,intraperitoneal, intranasal, transdermal, rectal, oral, or buccalroutes. Parenteral administration can be by bolus injection or bygradual perfusion over time. Preparations for parenteral administrationinclude sterile aqueous or non-aqueous solutions, suspensions, andemulsions, which may contain auxiliary agents or excipients known in theart, and can be prepared according to routine methods. In addition,suspension of the active compounds as appropriate oily injectionsuspensions may be administered. Suitable lipophilic solvents orvehicles include fatty oils, for example, sesame oil, or synthetic fattyacid esters, for example, sesame oil, or synthetic fatty acid esters,for example, ethyl oleate or triglycerides. Aqueous injectionsuspensions that may contain substances increasing the viscosity of thesuspension include, for example, sodium carboxymethyl cellulose,sorbitol, and/or dextran. Optionally, the suspension may also containstabilizers. Pharmaceutical compositions include suitable solutions foradministration by injection, and contain from about 0.01 to 99 percent,preferably from about 20 to 75 percent of active compound together withthe excipient. Compositions which can be administered rectally includesuppositories.

It is understood that the dosage administered will be dependent upon theage, sex, health, and weight of the recipient, kind of concurrenttreatment, if any, frequency of treatment, and the nature of the effectdesired. The dosage will be tailored to the individual subject, as isunderstood and determinable by one of skill in the art. The total doserequired for each treatment may be administered by multiple doses or ina single dose. The pharmaceutical composition of the present inventionmay be administered alone or in conjunction with other therapeuticsdirected to the condition, or directed to other symptoms of thecondition. Usually a daily dosage of active ingredient is comprisedbetween 0.01 to 100 milligrams per kilogram of body weight. Ordinarily 1to 40 milligrams per kilogram per day given in divided doses or insustained release form is effective to obtain the desired results.Second or subsequent administrations can be performed at a dosage, whichis the same, less than, or greater than the initial or previous doseadministered to the individual.

The present invention has been described with reference to the specificembodiments, but the content of the description comprises allmodifications and substitutions, which can be brought by a personskilled in the art without extending beyond the meaning and purpose ofthe claims.

The invention will now be described by means of the following Examples,which should not be construed as in any way limiting the presentinvention. The Examples will refer to the Figures specified here below.

EXAMPLES Example 1 In Vitro Characterization of the Non-heparin BindingMCP-1 Mutant MCP-1WT*2A

Materials and Methods

Expression of the MCP-1 Mutants MCP-1WT* and MCP-1WT*2A.

MCP-1 mutants were generated by in vitro, PCR-based mutagenesis of theDNA sequence coding for human MCP-1 (hMCP-1; FIG. 1; SEQ ID NO: 1), andin particular for the mature form of human MCP-1, corresponding to thesegment 24-99 of the precursor molecule (SWISSPROT Acc. No P13500).

It was first generated a plasmid encoding for an active mutant MCP-1protein called MCP-1WT*, in which a Methionine start codon is added atthe N-terminal of sequence coding for human MCP-1 (24-99) and aninternal Methionine (amino acid 87 in the precursor and 64 in the matureprotein) is replaced with an Isoleucine. This substitution avoids, whenthe plasmid is used for the expression in E. coli, the formation ofundesirable MCP-1 species containing Methionine Sulfoxide, withoutaffecting the properties typical of MCP-1 (Paavola C D et al, 1998). Theresulting sequence, called MCP-1WT*, was cloned and expressed in E. coliby making use of a plasmid based on the pET3 plasmid (Paavola C D et al,1998) as a protein containing 77 residues (FIG. 1A; SEQ ID NO: 2).

The plasmid expressing MCP-1WT* was then further mutagenized by cloninga PCR fragment encoding for two Alanines instead of Arginine and Lysinein positions 41 and 42 of human MCP-1 precursor (position 18 and 19 inthe mature protein), in order to generate the MCP-1 mutant MCP-1WT*2A,having the same length and purification features of MCP-1WT* (FIG. 1;SEQ ID NO: 3).

All constructs were obtained and controlled by standard molecularbiology technologies (PCR mutagenesis and amplification, DNA sequencing,restriction digestion) and maintained in the DH5alpha strain of E. coliduring the cloning process. The coding sequences were chosen in order tohave an optimal codon usage for the expression in E. coli i (Kane J F,1995).

The pET3-based plasmids encoding for MCP-1WT* and MCP-1WT*2A were thentransferred in an E. coli BL21 (pLys)-derived strain called TAP302 andthe resulting strains were used the recombinant expression of the MCP-1mutants as described (Paavola C D et al, 1998). This protocol includesthe use of aminopeptidase to remove the N-terminal methionine, thusobtaining recombinant MCP-1 mutants having the same length of thenatural mature form (76 amino acids; FIG. 1B). The identity of therecombinant proteins was verified by mass spectrometry.

Chromatographic assays of MCP-1WT* and MCP-1WT*2A.

MCP-1WT* and MCP-1WT*2A were loaded either onto a Heparin Sepharosecolumn or onto a SP Sepharose cation exchange column. In both cases thecolumn was equilibrated in 10 mM potassium phosphate (pH 7.5) and theprotein was eluted with a linear gradient from 0 to 1 M NaCl in the samebuffer.

Heparin Binding Assay of MCP-1WT* and MCP-1WT*2A.

Serial dilutions of MCP-1WT* mutants in Phosphate Buffer Saline (PBS)covering the range of 0.02-30 μM were incubated with 170 nM of[³H]-heparin for 1 hour at 37° C. Triplicates of 20 μl of each samplewere transferred to a 96 well P81 Unifilter plate (Whatman Inc) fittedwith a nitrocullose filter. The plate was washed three times with 200 μlof PBS using a vacuum pump to remove unbound labelled heparin.Scintillation fluid (50 μl) was added to each well and radioactivitycounted (1 minute/well) in a beta counter. Data were analysed usingPrism program (GraphPad Software).

Equilibrium Competition Receptor Binding Assays

The assays were carried out on membranes from Chinese Hamster Ovary(CHO) cells stably expressing the MCP-1 receptor (CCR2), using aScintillation Proximity Assay (SPA) and [¹²⁵I]-MCP-1 as tracer. Theradiolabelled chemokine (specific activity of 2200 mCi/mole) wasgenerated from recombinant mature MCP-1 according to the [¹²⁵I] supplier(Amersham). Competitors were prepared by serial dilutions (range10⁻⁶-10⁻¹² M) of the unlabelled MCP-1 mutant in the binding buffer (50mM HEPES pH 7.2, 1 mM CaCl₂, 5 mM MgCl₂, 0.15 M NaCl and 0.5% BSA).

Wheat germ SPA beads (Amersham) were suspended in PBS to 50 mg/ml, anddiluted in the binding buffer to a 10 mg/ml, and the final concentrationin the assay was 0.25 mg/well. Membranes expressing CCR2 were stored at−80° C. and diluted in the binding buffer to 80 μg/ml. The finalmembrane concentration was 2 μg/well and that of [¹²⁵I]-MCP-1 was 0.1nM. The plates were incubated at room temperature with agitation for 4hours. Radioactivity was counted (1 minute/well) in a beta counter. Datafrom triplicate samples were analysed using Grafit program (ErithacusSoftware).

Results

The literature shows different approaches for testing the heparin/GAGbinding properties of MCP-1 (Hoogewerf A J et al., 1997; Kuschert G etal., 1999; Ali S et al., 2001; Patel D et al., 2001; Chakravarty L etal, 1998).

Two mutants of MCP-1, whose sequence is based on the mature form ofhuman MCP-1 (FIG. 1), were expressed in E. coli. The first one,MCP-1WT*, corresponds to mature human MCP-1 precursor with a mutationknown to eliminate the possibility of methionine oxidation, withoutinterfering with the binding properties and activity typical of MCP-1(Paavola C D et al, 1998). On the basis of the sequence of this “active”mutant, a second mutant, MCP-1WT*2A was expressed in which a dibasicsite at the N-terminal was additionally replaced with Alanine residues.

The effect of this latter substitution on the MCP-1 properties was firsttested by heparin chromatography. The elution profiles of the twomutants applied on this chromatography column differ considerably, sincethe concentration of NaCl required to elute MCP-1WT* was 0.54 M NaClwhilst MCP-1WT*2A was eluted at 0.24 M NaCl. A smaller difference wasmeasured using cation exchange chromatography on a SP Sepharose column(0.55 M NaCl against 0.27 M NaCl).

The comparison of the elution profiles on such chromatographic mediaprovides a qualitative indication on the contribution of non-specificelectrostatic interactions due to basic amino acids to the heparinbinding properties (Proudfoot A et al., 2001). The difference in NaClconcentration obtained on cation exchange chromatography is subtractedfrom that obtained on heparin chromatography. According to this method,when the resulting figure is positive (0.02 M, in this case), it can beconcluded that a specific interaction with heparin is identified asbeing associated to the dibasic site mutated in MCP-1WT*2A.

A direct measure of binding to heparin was then performed usingtritiated heparin and serial dilution of the E. coli expressed MCP-1mutants (FIG. 2). Protein-Heparin complexes were isolated by adding thedilutions onto nitrocellulose filters. Such supports are capable ofretaining proteins efficiently, therefore allowing the evaluation ofamount of the radiolabelled heparin bound to the protein. This approachconfirmed that the heparin binding properties of MCP-1WT*2A weresignificantly reduced compared to MCP-1WT*.

Finally, an equilibrium competition receptor binding assay was performedto demonstrate the effect of the reduced heparin binding properties ofMCP-1WT*2A on the binding of the specific receptor CCR2 (FIG. 3).Samples containing radiolabelled MCP-1 mixed with serial dilutions ofone of the two mutants, or of MCP-1, were incubated with membranesprepared from CHO cells stably expressing CCR2. Whilst MCP-1WT* andMCP-1 protein showed an almost identical binding profile, the MCP-1WT*2Amutant shows a 20 fold reduction in affinity for CCR2, since it has anIC₅₀ of 1.73±0.6 nM, compared to 0.08±0.045 nM for the other two testedproteins, showing that high affinity is retained in this heparin-bindingdefective MCP-1 mutant.

Example 2 Characterization of the Non-heparin Binding MCP-1 Mutant inModels of Cell Recruitment

Materials and Methods

Chemotaxis Assay

The assay was carried out using a human pro-monocytic cell line (THP-1)in 24-well transwell chemotaxis chambers (Costar) fitted with 5 μm poresize membranes (Neuroprobe). The recombinant MCP-1 proteins wereserially diluted (range of 10⁻⁶-10⁻¹² M) in 600 μl of RPMI mediumcontaining 5% inactivated fetal calf serum (FCS), 2 mM glutamine and 25mM HEPES (pH 7.2). These samples were placed in the lower wells, whilstTHP-1 cells (100 μl of a cell suspension at 10×10⁶ cells/ml in the samemedium) were placed in the inserts. The chamber was incubated for 3hours at 37° C. under 5% CO₂. The samples were then removed, transferredto a 1.5 ml tube, and centrifuged at 200×g for 5 minutes. The pelletedcells were resuspended in 100 ml PBS and counted in a Coulter counter(Beckman). The data were analyzed using Prism program (GraphPadSoftware).

Peritoneal Cellular Recruitment Assay

Cellular recruitment was induced by intraperitoneal injection of femaleBALB/c mice of 8 to 12 wk of age of 10 μg of the recombinant MCP-1WT* orMCP-1WT*2A proteins diluted in 0.2-ml sterile, lipopolysaccharide-freePBS. When the antagonistic properties of MCP-1WT*2A were tested, theindicated amounts of the protein, diluted in 0.2 ml of the same sterilesolution, were administered 30 minutes prior to the agonist (MCP-1WT*)administration. Mice were sacrificed by aerosolized CO₂ 16 hours afterthe administration of the agonist, and peritoneal lavage was performedwith 5 ml PBS three times. The lavages were pooled and centrifuged at600×g for 10 minutes, and the pelleted cells were resuspended in a finalvolume of 1 ml and total elicited leukocytes were counted with ahemacytometer.

Results

Cell recruitment assays have been used for characterizing properties ofdifferent proteins related to MCP-1 (Ajuebor M N et al., 1998; RecklessJ and Grainger D J, 1999; Kaji M et al., 2001)

The results obtained for MCP-1WT*2A in a chemotaxis assay on humanmonocytes (THP-1 cell line) correspond well with those obtained in thereceptor-binding assay described in Example 1. MCP-1WT*2A was able toinduce a robust response (6-fold over baseline) of THP-1 chemotaxisalthough maximum activity was observed at 10 nM compared to 1 nM for thewild type proteins, which induced a 9-fold increase over baseline (FIG.4).

The activity of MCP-1WT*2A as an antagonist or agonist of MCP-1 was alsoevaluated using a peritoneal cellular recruitment assay (FIG. 5). WhenMCP-1WT* and MCP-1WT*2A are administered, the heparin-binding defectivemutant was unable to induce cellular recruitment into the peritoneum atthe dose (10 micrograms/mouse) that MCP-1WT* causes substantialrecruitment. Furthermore, if MCP-1WT*2A is administered 30 minutes priorto the administration of MCP-1WT*, the cellular recruitment induced byMCP-1WT* is significantly antagonized in a dose dependent manner.Therefore, the abrogation of GAG-binding in MCP-1 produces an antagonistof MCP-1 capable of inhibiting in vivo the cellular recruitment inducedby MCP-1.

Example 3 Characterization of a Non-heparin Binding MCP-1 Mutant inAnimal-based Models

Materials and Methods

Delayed Contact Hypersensitivity Model

The mouse ear-swelling test to measure contact hypersensitivity wasperformed as previously described (Garrigue J L et al., 1994). Briefly,mice were pre-sensitized topically by applying 25 μl of 0.5%2,4-dinitrofluorobenzene (DNFB; Sigma Chemical Co.) solution inacetone/olive oil (4:1) to the shaved abdomen. Five days later, 20 μl of0.2% DNFB in the same vehicle was applied to the right ears, and vehiclealone to the left ears. Mice were treated daily from Day 5 to 9 with anintraperitoneal administration of either 0.5 mg/kg (10 micrograms/mouse)of MCP-1WT* or PBS only in the control group. The first treatment wasadministered 1 hour prior to the DNFB challenge. Ear thickness wasmeasured with a dial thickness gauge (Mitutoyo Corp.), and ear swellingwas estimated by subtracting the pre-challenge from the post-challengevalue, and by further subtracting any swelling detected in thevehicle-challenged contralateral ear.

Bleomycin Induced Lung Fibrosis Model

C57BU6 female mice received bleomycin (3.75 U/kg in 25 μl PBS)intra-tracheally (day 0). One hour after the instillation of bleomycin,test animals received intraperitoneally either 0.25 mg/kg MCP-1WT*2A in0.2 ml PBS or only 0.2 ml PBS. This treatment was given daily andcontinued for 10 days. The body weight loss and percentage of mortalitywere recorded daily. At day 10, all mice were sacrified by CO ₂asphyxiation. Four lung lobes were placed at −80° C. for measurement ofhydroxyproline levels as an indication of collagen deposition as well asone lobe processed for histological determination of pulmonary fibrosis.Total lung collagen was determined by the analysis of hydroxyproline.Briefly, lungs were homogenized in Tris-HCl (pH 7.6) with a TissueTearor followed by incubation in Amberlite overnight at 115° C.Citrate/acetate buffer, isopropanol, chloramine-T and DAB solutions wereadded to the samples and left for 30 minutes at 60° C. Samples werecooled at room temperature for 10 minutes and read at 560 nm onspectrophotometer. Pulmonary fibrosis was also determined histologicallyby fixation of the right lung lobe in 10% Formalin, followed byembedding in paraffin, sectioning, and staining with Masson's trichromesolution. Histological changes were examined by light microscopy.Morphological evaluation of bleomycin-induced lung inflammation andfibrosis was performed using a semi-quantitative scoring method,calculating the percentage of the fibrotic area.

Experimental Autoimmune Encephalomyelitis (EAE) Model

Female mice (8-week old; C57 BU6NCrIBR strain; 18-22 grams of weight)were immunized at day 0 by injecting 0.2 ml of an emulsion containingthe MOG₃₅₋₅₅ peptide (200 micrograms) and Mycobacterium tuberculosis(500 micrograms) in Complete Freund's Adjuvant (CFA; Difco Lab.)subcutaneously in the left flank. Immediately afterwards, pertussistoxin (500 nanograms in 400 microliters of a buffer containing 0.5 MNaCl, 15 mM Tris (pH 7.5), 0.017% Triton X-100) was administeredintraperitoneally. On day 2 the animals were given a secondintraperitoneal injection of the same solution containing pertussistoxin. On day 7, the mice were administered a second dose of MOG₃₅₋₅₅peptide (200 micrograms) in CFA injected subcutaneously in the rightflank. This procedure results in disease onset at approximately day18-20, with the appearance of a progressive paralysis, arising from thetail and progressively ascending up to the forelimbs.

The treatment was started for each animal at experimental day 7(approximately 1-3 days before the usual occurrence of the disease) andcontinued for 21 consecutive days. Starting from day 7, the animals wereexamined individually for the presence of paralysis by means of aclinical score as follows:

0=no sign of disease

0.5=partial tail paralysis

1=tail paralysis

1.5=tail paralysis+partial unilateral hindlimb paralysis

2=tail paralysis+hindlimb weakness or partial hindlimb paralysis

2.5=tail paralysis+partial hindlimb paralysis (lowered pelvis)

3=tail paralysis+complete hindlimb paralysis

3.5=tail paralysis+complete hindlimb paralysis+incontinence

4=tail paralysis+hindlimb paralysis+weakness or partial paralysis offorelimbs

5=moribund or dead

The experiment was carried out with three groups of 10 animals each, asindicated below. Group 1 was treated with PBS as the positive control.Group 2 was treated daily with an intraperitoneal injection of 0.05mg/kg of MCP-1WT*2A. Group 3 was treated daily with an intraperitonealinjection of 0.5 mg/kg of MCP-1WT*2A. The MCP-1WT*2A mutant protein wasdissolved in sterile water and then diluted into sterile PBS (200microliters/mouse) to attain the required concentration.

Collagen Induced Arthritis (CIA) Model

Male mice (8-12 week old; DBA/1 strain; 18-22 grams of weight) wereimmunized at day 0 by intradermal injection at the base of the tail with0.2 ml of an emulsion composed of bovine type II collagen (100micrograms; Morwell Diagnostics) and Mycobacterium tuberculosis (400micrograms) in Complete Freund's Adjuvant (CFA; Difco Lab.).

Starting approximately from day 16-20, the signs of inflammationappeared, affecting one or more limbs. The animals were gradedindividually for disease severity by means of a clinical score, based onvisual clinical score for the presence of inflammation in the fingers ofthe forepaws and hind paws, and composed as follows:

0=no sign of disease

0.5=from 1 to 5 fingers/toes with signs of inflammation

1=from 6 to 10 fingers/toes with signs of inflammation

1.5=from 11 to 15 fingers/toes with signs of inflammation

2=from 16 to 20 fingers/toes with signs of inflammation

Two groups (n=10 mice) having a total clinical score >0.5 were dailytreated for 7 consecutive days with intraperitoneal injections of PBS(control group) or with 0.05 mg/kg of MCP-1WT*2A. All the animals weresacrificed 24 hours after the last treatment.

Ovalbumin Induced Lung Inflammation (OVA) Model

Female mice (8-10 weeks old; Balb/c strain). Mice were sensitized by anintraperitoneal injection of 10 μg ovalbumin (Sigma) precipitated in 2mg aluminium hydroxide 2% (Serva) in a total volume of 200 μl. Thealuminium hydroxide 2% /ovalbumin solution was prepared by mixing 25 μlovalbumin (2 mg/ml), 250 μl aluminium hydroxide in 725 μl LPS-free 0.9%NaCl and precipitated 3-4 hours at 4° C. Fifteen days aftersensitization, mice were treated and challenged in groups of 6 mice asfollows:

Group 1: challenged with LPS-free 0.9% NaCl and treated with PBS(baseline)

Group 2: challenged with ovalbumin and treated with PBS (negativecontrol)

Group 3: challenged with ovalbumin and treated with 0.5 mg/kg MCP-1WT*2A

PBS or MCP-1WT*2A were administered by intraperitoneal injection (200microliters) 30 minutes before each challenge on five consecutive days.Mice were challenged intranasally, with ovalbumin (15 micrograms ofprecipitated ovalbumin resusped in 50 microliters LPS-free 0.9% NaCl)under inhaled anaesthesia with Isoflurane. At 72 hours post-challenge,mice were killed by a lethal intraperitoneal injection of 300 μl 14%Urethane (v:v) in 0.9% NaCl. The trachea was trimmed free of connectivetissue, and a small incision was made to insert a catheter of 0.75millimeters diameter into the trachea. The cannula was tied in placewith a piece of suture thread and was attached to 1-ml syringe. Lungswere filled in situ with 0.4 millililter PBS. Fluid was withdrawn fromthe lungs after gentle massage to remove cells and collected in aplastic tube on ice. This procedure was repeated 4 times and the cellsuspensions recovered from each animal were combined on ice to give afinal volume of approximately 1.4 ml. Cell counting was performed usinga hemacytometer using a 2-fold dilution of the cell suspension intoTrypan blue.

Results

The in vivo properties of MCP-1 have been characterized in manyarticles, in particular by making use of transgenic mice (Lu B et al.,1998; Rutledge B J et al., 1995). MCP-1WT*2A were then tested by makinguse of animal models for human inflammation and diseases to confirm theresults on the antagonistic activity of this MCP-1 heparin-bindingdefective mutant obtained using the peritoneal cell recruitment model.

Delayed contact hypersensitivity is an animal model in which ahapten-specific skin inflammation mediated by T cells is measured by earswelling. Enhanced contact hypersensitivity has been shown in transgenicmice that constitutively produce high levels of MCP-1 in the sera(Mizumoto N et al., 2001). The ear skin of normal mice was challenged bymaking use of the contact sensitizer 2,4-dinitrofluorobenzene (DNFB) ashapten. The consequent swelling was significantly lower in mice treatedwith an intraperitoneal administration of MCP-1WT*2A (starting at thetime of challenge with DNFB), when compared to the effect observed inmice treated with vehicle alone, throughout the treatment period (FIG.6).

The properties of MCP-1WT*2A were tested in a lung inflammation/fibrosismodel, since it is known that MCP-1 induces procollagen deposition inpulmonary or skin inflammatory processes (Hogaboam C M et al., 1999).

Intratracheal instillation of bleomycin in mice results in lunginflammation and fibrosis within 7 to 10 days respectively, with amarked accumulation of collagen in the lungs as well as a rapid decreaseof weight (Chen E S et al., 2001). Weight was recorded throughout the 10days after the exposure to bleomycin in mice treated 1 hour later withan intraperitoneal administration of MCP-1WT*2A or only with PBS, andfurther compared to a control group not treated with bleomycin. As it isclearly evident starting from day 2, PBS-treated control mice lose asignificantly higher amount of weight compared to MCP-1WT*2A treatedmice (FIG. 7). Lung fibrosis and inflammation was evaluated aftersacrificing the animals at day 10 using two different methods. It isknown that bleomycin increases lung hydroxyproline synthesisproportionally to collagen synthesis and fibrosis (Madtes D K et al.,1999). The hydroxyproline level s measured in mice treated with PBS onlywere significantly higher than the levels measured in the groupreceiving an intraperitoneal administration of MCP-1WT*2A. Indeed, thelevels in this latter group were comparable to non-bleomycin treatedmice. Another semi quantitative histological assessment confirmed thesignificant reduction in total levels of fibrosis in the MCP-1WT*2Atreated group versus control (FIG. 8).

Antibodies against MCP-1 and transgenic mice lacking a functional MCP-1gene demonstrated the crucial role of MCP-1 for macrophage recruitmentand inflammation in central nervous system associated, for example, toHerpesvirus-Induced Encephalomyelitis (HSM) and Experimental AutoimmuneEncephalomyelitis (EAE), the animal model for multiple sclerosis(Nakajima H et al., 2001; Huang D R et al., 2001). The administration ofMCP-1WT*2A considerably improved the clinical score of EAE animal modelat both the tested doses (FIG. 9).

As discussed above, MCP-1 has a strong fibrogenic effect. The propertiesof MCP-1WT*2A against this MCP-1 activity were tested in theCollagen-induced Arthritis (CIA) model. Also in this case, MCP-1WT*2Aconsiderably improved the clinical score of the treated mice (FIG. 10).

Lung allergic inflammation and bronchial hyperresponsiveness (BHR) thatcharacterize asthma is achieved by the accumulation and activation ofdifferent leukocyte subsets in the lung. Blockage of MCP-1 by means ofantibodies diminishes drastically BHR and inflammation (Gonzalo J A etal., 1998). In the ovalbumin-induced lung inflammation (OVA) model,MCP-1WT*2A provided significant improvement, in terms of reduced cellrecruitment (FIG. 11).

Given that the dibasic site mutated in the examples of the presentinvention, together with the other residues known to be involved inMCP-1 binding to GAG such as Histidine 66 and Lysine 58 (Chakravarty Let al., 1999), is conserved in all MCPs (FIG. 1B), other MCPs-basedmutants having antagonistic activities can be designed on the basis ofthe findings of this patent application.

In particular, MCP antagonists can be double mutants of human matureMCP-1 (SEQ ID NO: 4), MCP-2 (SEQ ID NO: 5), MCP-3 (SEQ ID NO: 6), MCP-4(SEQ ID NO: 7), or Eotaxin (SEQ ID NO: 8) in the positions 18 and 19, 18and 58 (or 66), 19 and 58 (or 66), as well as triple mutants in thepositions 18, 19 and 58 (or 66; the numbering corresponds the one givenfor human mature MCP-1). Other residues that can be mutated additionallyto the ones in positions 18 and 19 are the other basic residuesidentified as highly conserved in all human MCP proteins (residues 24,44, 49, 75; FIG. 1B).

The properties of these alternative molecules can be tested by any ofthe methods above described, as well as by making use of othervalidating approaches known in the art. Many other usefulchemokine-related technologies (recombinant expression, in vitro assays,transgenic animals) are extensively reviewed in literature (“ChemokineProtocols”, Methods in Molecular Biology vol. 138, Humana Press, 2000;“Chemokine Receptors”, Methods in Enzymology vol. 288. Academic Press,1997).

TABLE I Amino More Preferred Acid Synonymous Group Synonymous Groups AlaGly, Thr, Pro, Ala, Ser Gly, Ala Arg Asn, Lys, Gln, Arg, His Arg, Lys,His Asn Glu, Asn, Asp, Gln Asn, Gln Asp Glu, Asn, Asp, Gln Asp, Glu CysSer, Thr, Cys Cys Gln Glu, Asn, Asp, Gln Asn, Gln Glu Glu, Asn, Asp, GlnAsp, Glu Gly Ala, Thr, Pro, Ser, Gly Gly, Ala His Asn, Lys, Gln, Arg,His Arg, Lys, His Ile Phe, Ile, Val, Leu, Met Ile, Val, Leu, Met LeuPhe, Ile, Val, Leu, Met Ile, Val, Leu, Met Lys Asn, Lys, Gln, Arg, HisArg, Lys, His Met Phe, Ile, Val, Leu, Met Ile, Val, Leu, Met Phe Trp,Phe, Tyr Tyr, Phe Pro Gly, Ala, Ser, Thr, Pro Pro Ser Gly, Ala, Ser,Thr, Pro Thr, Ser Thr Gly, Ala, Ser, Thr, Pro Thr, Ser Trp Trp, Phe, TyrTrp Tyr Trp, Phe, Tyr Phe, Tyr Val Met, Phe, Ile, Leu, Val Met, Ile,Val, Leu

TABLE II Amino Acid Synonymous Group Ala D-Ala, Gly, Aib, B-Ala, Acp,L-Cys, D-Cys Arg D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met, Ile,D-.Met, D-Ile, Orn, D-Orn Asn D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-GlnAsp D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cys D-Cys, S—Me-Cys, Met,D-Met, Thr, D-Thr Gln D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp GluD-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Gly Ala, D-Ala, Pro, D-Pro,Aib, .beta.-Ala, Acp Ile D-Ile, Val, D-Val, AdaA, AdaG, Leu, D-Leu, Met,D-Met Leu D-Leu, Val, D-Val, AdaA, AdaG, Leu, D-Leu, Met, D-Met LysD-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn,D-Orn Met D-Met, S—Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val Phe D-Phe,Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp, Trans-3,4, or5-phenylproline, AdaA, AdaG, cis-3,4, or 5-phenylproline, Bpa, D-Bpa ProD-Pro, L-I-thioazolidine-4-carboxylic acid, D-orL-1-oxazolidine-4-carboxylic acid Ser D-Ser, Thr, D-Thr, allo-Thr, Met,D-Met, Met(O), D-Met(O), L-Cys, D-Cys Thr D-Thr, Ser, D-Ser, allo-Thr,Met, D-Met, Met(O), D-Met(O), Val, D-Val Tyr D-Tyr, Phe, D-Phe, L-Dopa,His, D-His Val D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met, AdaA, AdaG

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1. An isolated MCP protein comprising: a) amino acid substitutions atpositions 18 and 19, as numbered on the sequence of human mature MCP-1,said human mature MCP-1 corresponding to amino acids 24-99 of SEQ ID NO:1, wherein amino acids at positions 18 and 19 are substituted withalanine, glycine, serine, threonine, proline, aspartic acid, asparagine,glutamic acid or glutamine and wherein said MCP protein antagonizes anactivity of unaltered MCP proteins; or b) amino acid substitutions atpositions 18 and 19 and amino acid substitutions at one or more aminoacid positions numbered 24, 44, 49, 58, 66 and 75, as numbered on thesequence of human mature MCP-1, said human mature MCP-1 corresponding toamino acids 24-99 of SEQ ID NO: 1, wherein amino acids at positions 18and 19 are substituted with alanine, glycine, serine, threonine,proline, aspartic acid, asparagine, glutamic acid or glutamine andwherein said MCP protein antagonizes an activity of unaltered MCPproteins.
 2. The isolated MCP protein according to claim 1, wherein saidMCP protein comprises amino acid substitutions at positions 18 and 19and amino acid substitutions at one or more amino acid positionsnumbered 24, 44, 49, 58, 66 and 75, as numbered on the sequence of humanmature MCP-1, said human mature MCP-1 corresponding to amino acids 24-99of SEQ ID NO: 1, wherein amino acids at positions 18 and 19 aresubstituted with alanine, glycine, serine, threonine, proline, asparticacid, asparagine, glutamic acid or glutamine and wherein said MCPprotein antagonizes an activity of unaltered MCP proteins.
 3. Theisolated MCP protein according to claim 2, wherein said one or moreamino acid positions are substituted with alanine, glycine, serine,threonine, proline, aspartic acid, asparagine, glutamic acid orglutamine.
 4. The isolated MCP protein according to claim 1, whereinsaid MCP protein comprises amino acid substitutions at positions 18 and19, as numbered on the sequence of human mature MCP-1, said human matureMCP-1 corresponding to amino acids 24-99 of SEQ ID NO: 1, wherein aminoacids at positions 18 and 19 are substituted with alanine.
 5. Theisolated MCP protein according to claim 1, in which one amino acidresidue has been added, deleted, or substituted without interfering withthe antagonistic activity of said MCP protein with respect to unalteredMCP proteins.
 6. The isolated MCP protein according to claim 1, furthercomprising a heterologous amino acid sequence.
 7. The isolated MCPprotein according to claim 1, wherein said MCP protein is human MCP-1,human MCP-2, human MCP-3, human MCP-4, or human Eotaxin.
 8. The isolatedMCP protein according to claim 1, comprising the sequence of SEQ ID NO:3.
 9. The isolated MCP protein according to claim 1, further comprisinga molecule chosen from radioactive labels, biotin, fluorescent labels,cytotoxic agents, or drug delivery proteins.
 10. The isolated MCPprotein according to claim 6, wherein the heterologous amino acidsequence is selected from: extracellular domains of membrane-boundprotein, immunoglobulin constant regions, multimerization domains,extracellular proteins, signal peptide-containing proteins or exportsignal-containing proteins.
 11. The isolated MCP protein according toclaim 1, wherein said activity is the recruitment of leukocytes.
 12. Theisolated MCP protein according to claim 2, wherein said activity is therecruitment of leukocytes.
 13. The isolated MCP protein according toclaim 4, wherein said activity is the recruitment of leukocytes.
 14. Anisolated nucleic acid encoding a MCP protein comprising: a) amino acidsubstitutions at positions 18 and 19, as numbered on the sequence ofhuman mature MCP-1, said human mature MCP-1corresponding to amino acids24 -99 of SEQ ID NO: 1, wherein amino acids at positions 18 and 19 aresubstituted with alanine, glycine, serine, threonine, proline, asparticacid, asparagine, glutamic acid or glutamine and wherein said MCPprotein antagonizes an activity of unaltered MCP proteins; or b) aminoacid substitutions at positions 18 and 19 and amino acid substitutionsat one or more amino acid positions numbered 24, 44, 49, 58, 66 and 75,as numbered on the sequence of human mature MCP-1, said human matureMCP-1 corresponding to amino acids 24 -99 of SEQ ID NO: 1, wherein aminoacids at positions 18 and 19 are substituted with alanine, glycine.serine, threonine, proline, aspartic acid, asparagine, glutamic acid orglutamine and wherein said MCP protein antagonizes an activity ofunaltered MCP proteins.
 15. An expression vector comprising a nucleicacid encoding a MCP protein comprising: a) amino acid substitutions atpositions 18 and 19, as numbered on the sequence of human mature MCP-1,said human mature MCP-1 corresponding to amino acids 24 -99 of SEQ IDNO: 1, wherein amino acids at positions 18 and 19 are substituted withalanine, glycine, serine, threonine, proline, aspartic acid, asparagine,glutamic acid or glutamine and wherein said MCP protein antagonizes anactivity of unaltered MCP proteins; or b) amino acid substitutions atpositions 18 and 19 and amino acid substitutions at one or more aminoacid positions numbered 24, 44, 49, 58, 66 and 75, as numbered on thesequence of human mature MCP-1, said human mature MCP-1 corresponding toamino acids 24 -99 of SEQ ID NO: 1, wherein amino acids at positions 18and 19 are substituted with alanine, glycine, serine, threonine,proline, aspartic acid, asparagine, glutamic acid or glutamine andwherein said MCP protein antagonizes an activity of unaltered MCPproteins.
 16. An isolated host cell transformed with an expressionvector comprising a nucleic acid encoding a MCP protein comprising: a)amino acid substitutions at positions 18 and 19, as numbered on thesequence of human mature MCP- 1, said human mature MCP- 1 correspondingto amino acids 24 -99 of SEQ ID NO: 1, wherein amino acids at positions18 and 19 are substituted with alanine, glycine, serine, threonine,proline, aspartic acid, asparagine, glutamic acid or glutamine andwherein said MCP protein antagonizes an activity of unaltered MCPproteins; or b) amino acid substitutions at positions 18 and 19 andamino acid substitutions at one or more amino acid positions numbered24, 44, 49, 58, 66 and 75, as numbered on the sequence of human matureMCP-1, said human mature MCP-1 corresponding to amino acids 24 -99 ofSEQ ID NO: 1, wherein amino acids at positions 18 and 19 are substitutedwith alanine, glycine, serine, threonine, proline, aspartic acid,asparagine, glutamic acid or glutamine and wherein said MCP proteinantagonizes an activity of unaltered MCP proteins.
 17. A process ofpreparing a MCP antagonist comprising culturing a host cell transformedwith an expression vector comprising a nucleic acid encoding a MCPprotein comprising: a) amino acid substitutions at positions 18 and 19,as numbered on the sequence of human mature MCP-1, said human matureMCP-1corresponding to amino acids 24 -99 of SEQ ID NO: 1, wherein aminoacids at positions 18 and 19 are substituted with alanine, glycine,serine, threonine, proline, aspartic acid, asparagine, glutamic acid orglutamine and wherein said MCP protein antagonizes an activity ofunaltered MCP proteins; or b) amino acid substitutions at positions 18and 19 and amino acid substitutions at one or more amino acid positionsnumbered 24, 44, 49, 58, 66 and 75, as numbered on the sequence of humanmature MCP-1, said human mature MCP-1corresponding to amino acids 24 -99of SEQ ID NO: 1, wherein amino acids at positions 18 and 19 aresubstituted with alanine, glycine, serine, threonine, proline, asparticacid, asparagine, glutamic acid or glutamine and wherein said MCPprotein antagonizes an activity of unaltered MCP proteins.
 18. Acomposition comprising a carrier and a MCP protein comprising: a) aminoacid substitutions at positions 18 and 19, as numbered on the sequenceof human mature MCP-1, said human mature MCP-1corresponding to aminoacids 24 -99 of SEQ ID NO: 1, wherein amino acids at positions 18 and 19are substituted with alanine, glycine, serine, threonine, proline,aspartic acid, asparagine, glutamic acid or glutamine and wherein saidMCP protein antagonizes an activity of unaltered MCP proteins; or b)amino acid substitutions at positions 18 and 19 and amino acidsubstitutions at one or more amino acid positions numbered 24, 44, 49,58, 66 and 75, as numbered on the sequence of human mature MCP-1, saidhuman mature MCP-1corresponding to amino acids 24 -99 of SEQ ID NO: 1,wherein amino acids at positions 18 and 19 are substituted with alanine,glycine, serine, threonine, proline, aspartic acid, asparagine, glutamicacid or glutamine and wherein said MCP protein antagonizes an activityof unaltered MCP proteins.
 19. The composition according to claim 18,wherein said MCP protein comprises amino acid substitutions at positions18 and 19 and amino acid substitutions at one or more amino acidpositions numbered 24, 44, 49, 58, 66 and 75, as numbered on thesequence of human mature MCP-1, said human mature MCP-1 corresponding toamino acids 24 -99 of SEQ ID NO: 1, wherein amino acids at positions 18and 19 are substituted with alanine, glycine, serine, threonine,proline, aspartic acid, asparagine, glutamic acid or glutamine andwherein said MCP protein antagonizes an activity of unaltered MCPproteins.
 20. The composition according to claim 19, wherein said MCPprotein comprises amino acid substitutions at positions 18 and 19, asnumbered on the sequence of human mature MCP-1, said human matureMCP-1corresponding to amino acids 24 -99 of SEQ ID NO: 1, wherein aminoacids at positions 18 and 19 are substituted with alanine.
 21. Thecomposition according to claim 19, wherein said MCP protein comprisesamino acid substitutions at positions 18 and 19, as numbered on thesequence of human mature MCP-1, said human mature MCP-1corresponding toamino acids 24 -99 of SEQ ID NO: 1, wherein amino acids at positions 18and 19 are substituted with alanine, glycine, serine, threonine,proline, aspartic acid, asparagine, glutamic acid or glutamine.
 22. Thecomposition according to claim 18, wherein said MCP protein furthercomprises one amino acid residue that has been added, deleted, orsubstituted without interfering with the antagonistic activity of saidMCP protein.
 23. The composition according to claim 18, wherein said MCPprotein further comprises a heterologous amino acid sequence.
 24. Thecomposition according to claim 18, wherein said MCP protein is humanMCP-1, human MCP-2, human MCP-3, human MCP-4, or human Eotaxin.
 25. Thecomposition according to claim 18, wherein said MCP protein comprisesSEQ ID NO:
 3. 26. The composition according to claim 18, wherein saidMCP protein further comprises a molecule chosen from radioactive labels,biotin, fluorescent labels, cytotoxic agents, or drug delivery proteins.27. The composition according to claim 23, wherein the heterologousamino acid sequence is selected from: extracellular domains ofmembrane-bound protein, immunoglobulin constant regions, multimerizationdomains, extracellular proteins, signal peptide-containing proteins orexport signal-containing proteins.
 28. The composition according toclaim 18, wherein said MCP protein has amino acids at positions 18 and19 substituted with alanine.